Highlights Of The Chapter

• Optical bioimaging can be used to investigate structures and functions of cells and tissues and to profile diseases at cellular, tissue, and in vivo specimen levels.

• Fluorescence microscopy, a preferred method for cellular imaging, is also useful for ex vivo and in vivo tissue imaging. Both endogenous and exogenous fluorophores are useful for fluorescence bioimaging. Exogenous fluorophores can be used as such or chemically conjugated to target a specific organelle to be imaged.

• An ideal fluorophore for bioimaging has (i) dispersability in the biological medium to be probed, (ii) specific association with a target molecule, organelle, or cell, (iii) high quantum efficiency of emission, (iv) environmental stability, and (v) the absence of photobleaching.

• In addition to the usual fluorescent organic dyes, some other bioimaging fluorophores are: organometallic complexes, near-IR and IR fluo-rophores, highly efficient new two-photon absorbing fluorophores, and inorganic nanoparticle emitters.

• Another important class of fluorescent labels is fluorescent proteins (FP), which exhibit some unique features like resistance to denaturation and ease in coupling to other biomolecules. The fluorescent proteins are expressed in the cell and thus generated in situ by introducing an FP gene in a living organism.

• A wide variety of mutant variants of fluorescent proteins, producing fluorescence of varying wavelengths covering the entire visible spectral range and with enhanced emission, are available. They can be used for a wide range of applications such as a cell lineage tracer, reporter for gene expression or a measure of protein-protein interactions.

• Using selective fluorescent labels, bioimaging of a specific organelle can be accomplished to study its structure and function. An example is selective staining of mitochondria by a dye Mitotracker to monitor cell apotosis and measure mitochondrial membrane potential.

• Both confocal and near-field microscopy have been used for imaging of microbes such as viruses and bacteria.

• Near-field microscopy (NSOM) provides the resolution of <100 nm needed to study the structural details of viruses and bacteria. However, their applications to image viruses and bacteria have been limited, because traditional NSOM requires fixing them and they are not in their natural environment. Examples of NSOM images are that of a tobacco mosaic virus and a Porphyromonas gingivalis oral bacteria.

• Cellular imaging can be used to probe a cell's ionic environment, measure intracellular pH measurements, monitor drug-cell interactions, and determine nucleic acid distribution.

• Cellular imaging by two-photon laser scanning microscopy using a fluo-rescently labeled chemotherapeutic drug-carrier conjugate can optically track the cellular pathway and clarify the drug's mechanism of action. The bioimaging, together with localized (site-specific) fluorescence spectroscopy, indicates that the chemotherapeutic activity of the drug, doxorubicin, is due to its ability to intercalate into DNA and break the strands of the double helix by inhibiting topoisomerase II.

• Examples for confocal imaging of nucleic acid distribution and differentiation of RNA-DNA content are given. Some fluorescent probes are specific to double-stranded DNA, while others bind to both double- and single-stranded nucleic acids.

• Fluorescence in situ hybridization (FISH) enables the detection and determination of the spatial distribution of specific DNA or RNA sequences in the cytoplasm, nucleus, and chromosomes. FISH involves in situ hybridization of nucleic acids in the target cells or chromosomes to be detected or imaged, with fluorescently labeled single-stranded probe nucleic acids.

• Cellular interactions, such as protein-protein interactions, can be probed by fluorescent resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM). A suitable energy donor and acceptor pair involves cyanofluorescent protein and yellow fluorescent protein.

• Tissue imaging using optical techniques can be achieved for soft and hard tissues under both ex vivo and in vivo conditions.

• Examples provided of imaging of soft tissues are (a) optical sectioning of a corneal tissue treated with a two-photon conjugated polyacrylic acid nanoparticles and (b) extracted tumor tissue of a hamster cheek pouch with selective accumulation of the drug: two-photon fluorophore conjugate. Both images are obtained by using two-photon laser scanning microscopy.

• Both two-photon laser scanning microscopy and optical coherence tomography have proved to be more suitable than other ways of optical bioimaging in highly scattering media such as hard tissues.

• Examples provided are (i) OCT images of a section of a human tooth displaying the ability to differentiate the various structures of a tooth and (ii) three-dimensional reconstruction of two-photon laser scanning images of dentinal tubules obtained by filling them with a dental bonding material containing a two-photon dye. A sharp image of dentinal tubules indicates a deep penetration by the dental bonding agent.

• In vivo imaging can be used at the level of tissue, organ, or entire live object (animal or human being).

• An example of optically sectioned corneal imaging with confocal microscopy provides evidence of irreversible deep stromal degeneration caused by long-term wearing of contact lenses.

• Another example of in vivo imaging utilizes GFP expressed in tumor cells to image tumor localization and growth in a live animal.

• Retinal imaging in angiography is an important example of in vivo imaging. It utilizes an infrared dye, indocyanine green, to image the structures in the back of the eye for finding leakage or damage to the blood vessels that nourish the retina.

• An exciting area of in vivo imaging is optical mammography, also known as laser mammography, for the noninvasive detection of breast cancer.

• A new approach in optical mammography is the use of a near-infrared dye administered in a self-quenched nonfluorescent state. The fluorescence of the dye is activated (restored) by an enzyme, overexpressed by tumors, thus enabling one to use fluorescence imaging for the study of tumor localization and growth.

• Catheter-based endoscopic optical coherence tomography (OCT) is emerging as a powerful approach for in vivo imaging of highly scattering tissues and organs. An application provided is gastrointestinal pathology.

• Future directions of research and development include (i) increased applications of near-IR imaging, thus opening opportunities for development of near-IR fluorphores and lasers, (ii) use of the nanoparticle approach for encapsulation and delivery to a specific biological size, (iii) in situ activation of a fluorescent probe in response to a stimulus or a drug, (iv) real-time in vivo imaging to monitor biological activities and (v) imaging of microbes much smaller than the wavelength of light in their natural environment.

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